| BackgroundRheumatoid arthritis(RA)is a chronic inflammatory autoimmune disease,which is characterized by symmetrical abnormal proliferation of synovial tissue and progressive joint destruction,and can also lead to functional impairment of various extra-articular tissues and organs,such as skin,blood vessels and kidneys.Autophagy plays an essential role in the pathogenesis of RA by influencing the antigen presentation of citrullinated protein,regulating the survival of T cells and B cells,inhibiting the apoptosis of synovial fibroblasts and promoting the proliferation of osteoclasts.At present,a large number of abnormally expressed lnc RNAs are recognized in the tissues and cells of RA patients,such as lnc RNA NEAT1,GAS5,H19,MEG3,MALAT1,etc.,but their mechanisms in the pathogenesis of RA need to be further explored.In view of the important role of Lnc RNA and micro RNA network in autophagy regulation,this study evaluated the correlation between lnc RNA and autophagy markers in RA patients,and found that the expression level of lnc RNA NEAT1 was correlated with RA autophagy markers.In view of ce RNA,SIRT1 is taken as the downstream target gene,and bioinformatics prediction shows that mi R-128-3p has binding sites with lnc RNA NEAT1 and SIRT1.Furthermore,through cellular functional experiments,the mechanism of Lnc RNA NEAT1/ mi R-128-3p/SIRT1 pathway in the regulation of RA autophagy was explored in this study,providing new clues for the prevention,diagnosis and treatment of RA.MethodsThis study was divided into four parts:(1)The correlation between the expression levels of candidate lnc RNA NEAT1,GAS5,H19,MEG3,MALAT1 and autophagy markers Beclin1 and LC3,and downstream target gene SIRT1 was evaluated by quantitative real-time polymerase chain reaction(q RT-PCR)through collecting peripheral blood mononuclear cell(PBMC)from RA patients and healthy controls.(2)The binding sites of mi R-128-3p with lnc RNA NEAT1 and SIRT1 were predicted by Starbase V3.0 database,mi Rcode database and DIANA-Lnc Base v2 database.(3)RA-FLS cell lines were resuscitated and cultured,and the expression of surface markers was detected by flow cytometry to identify the purity of RA-FLS cells.The expression levels of lnc RNA NEAT1,mi R-128-3p and SIRT1 in RA-FLS cells were altered by adenovirus and si RNA interference,and the expression of lnc RNA NEAT1,mi R-128-3p,SIRT1,Beclin1,LC3,IL-6 and MMP-3 in RA-FLS cells were detected by q RT-PCR to explore the mechanism of lnc RNA NEAT1/mi R-128-3p/SIRT1 pathway in RA autophagy regulation.(4)The relative expression level of luciferase in HEK 293 T cells transfected with NEAT1 and SIRT1 reporter gene vectors was detected by dual luciferase assay,so as to verify the binding between lnc RNA NEAT1 and mi R-128-3p,and mi R-128-3p and SIRT1.ResultsA total of 106 subjects were included in this study,including 47 RA petients and 59 healthy controls.RA patients were 52.1±12.4 years,and 39 RA patients(83.0%)were female,the healthy controls were 49.9±9.8 years old,and 51 healthy controls(86.4%)were female;and no significant difference was found in age and sex between the two groups(both P > 0.05).Flow cytometry showed that the CD90 positive rate of RA-FLS cells was 99.7%,which suggested that RA-FLS cells were of high purity and suitable for the cellular functional study.The q RT-PCR experiment showed that compared with healthy controls,the relative expression levels of Beclin1 and LC3 in RA patients were significantly higher(both P < 0.05);the relative expression levels of lnc RNA NEAT1,GAS5 and MALAT1 also increased significantly in RA patients(all P < 0.05),while the relative expression levels of H19 and MEG3 had no altered significantly(both P >0.05).Receiver operating characteristic curve found the Area Under Curve(AUC)of lnc RNA NEAT1,GAS5 and MALAT1 was 0.668,0.616 and 0.736,respectively(all P < 0.05),and the AUC of H19 and MEG3 was 0.552 and 0.492,respectively(both P > 0.05).Spearman correlation analysis found that the expression levels of lnc RNA NEAT1 and GAS5 in PBMC cells of RA patients were positively correlated with the expression level of Beclin1(both P < 0.05),while the expression levels of H19,MEG3 and MALAT1 were not significantly correlated with Beclin1(all P > 0.05);the expression levels of NEAT1,GAS5,H19 and MEG3 were positively correlated with the expression level of LC3(all P < 0.05),while the expression level of lnc RNA MALAT1 had no significant correlation with the expression level of LC3(P = 0.547).The q RT-PCR experiment further revealed that the expression level of SIRT1 in PBMC cells of RA patients was significantly higher than that of healthy controls(Z =-2.725,P = 0.006);the expression level of SIRT1 was positively correlated with the expression levels of lnc RNA NEAT1,MEG3 and MALAT1(all P < 0.05),but not with the expression levels of lnc RNA GAS5 and H19(both P > 0.05).On the basis of the above results,the target lnc RNA was identified as NEAT1 in this study,and then bioinformatic analysis and RA-FLS cell functional experiments were conducted.According to bioinformatics analysis,there are nine micro RNAs(mir-10a-5p,mi R-128-3p,mi R-206,mi R-212-3p,mi R-27a-3p,mi R-302 e,mi R-4500,mi R-490-3p,mi R-613)had competing binding sites with lnc RNA NEAT1,and other nine micro RNAs(mir-22-3p,mi R-199a-5p,mi R-128-3p,mi R-135a-5p,mi R-138-5p,mi R-141-3p,mi R-9-5p,mi R-200a-3p)had competing binding sites with SIRT1.Therefore,only mi R-128-3p has potential binding sites with both lnc RNA NEAT1 and SIRT1.Adenovirus/si RNA transfection of lnc RNA NEAT1 showed that NEAT1 adenovirus could significantly increase the expression levels of SIRT1,Beclin1 and LC3,IL-6 and MMP3,and inhibit the expression of mi R-128-3p in RA-FLS cells.Transfection with mi R-128-3p mimics/inhibitor showed that mi R-128-3p mimics could significantly down-regulate the expression levels of lnc RNA NEAT1,SIRT1,LC3,Beclin1,IL-6 and MMP-3 in RA-FLS cells.Furthermore,SIRT1adenovirus/si RNA transfection showed that SIRT1 adenovirus could significantly up-regulate the expression levels of LC3,Beclin1,IL-6 and MMP3 in RA-FLS cells.The double luciferase experiment revealed that mi R-128-3p mimics could significantly down-regulate the relative expression level of luciferase in HEK 293 T cells transfected with NEAT1 and SIRT1 wild-type reporter gene vectors,but does not change the relative expression level of luciferase in HEK 293 T cells transfected with NEAT1 and SIRT1 mutant reporter gene vectors.ConclusionIn this study,we found that the expression level of lnc RNA NEAT1 in RA patients was significantly increased,and significantly correlated with the expression levels of Beclin1 and LC3;there are binding sites of lnc RNA NEAT1 to both SIRT1 and mi R-128-3p;based on cell experiments,lnc RNA NEAT1 participates in the autophagy of RA-FLS cells and the secretion of cytokines such as IL-6 and MMP3 by competitively binding with mi R-128-3p and influencing the expression of SIRT1. |
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